WO2019039532A1 - Method for assessing risk of hepatocellular carcinoma - Google Patents

Abstract

Provided is a method for assessing risk of hepatocellular carcinoma, the method being rapid and simple and having high sensitivity and specificity. A method for assessing risk of hepatocellular carcinoma. The method includes: (1) a step for amplifying bisulfite-treated DNA originating from hepatic tissue of a subject, the DNA including a CpG site in an exon region of the MGRN1 gene; (2) a step for subjecting the obtained amplification product to ion exchange chromatography; and (3) a step for determining whether the DNA is DNA obtained from a subject having a high risk of onset of hepatocellular carcinoma on the basis of the shape of a peak in a detection signal of the chromatography.

Description

Risk assessment method for hepatocellular carcinoma

The present invention relates to a method for risk assessment of hepatocellular carcinoma using detection of methylated DNA.

Hepatocellular carcinoma (HCC) is a globally known malignancy, and its major developmental factor has been found to be hepatitis virus infection. The hepatitis viruses that cause hepatocellular carcinoma are mainly hepatitis B virus (HBV) and hepatitis C virus (HCV). HCC usually develops in patients suffering from chronic hepatitis or cirrhosis associated with hepatitis virus infection. Moreover, in most of the patients, since liver function has already deteriorated at the stage of onset of HCC, good treatment results can not be expected unless cancer is diagnosed early. Therefore, surveillance of premalignant conditions such as chronic hepatitis and cirrhosis should be given priority. In particular, patients at high risk of developing HCC should have close surveillance and early detection and treatment of HCC, even without subjective symptoms. On the other hand, close surveillance is an excessive burden for patients who do not have the risk of developing HCC. Therefore, for the appropriate management of patients with precancerous conditions such as chronic hepatitis and cirrhosis, the risk assessment of the development of HCC is very important.

Changes in DNA methylation are one of the most commonly observed epigenetic changes associated with carcinogenesis. Changes in DNA methylation have also been suggested to be involved in the early cancer and precancerous stages. It has been reported that changes in DNA methylation associated with aberrant splicing and / or expression of DNA methyltransferase occur in liver tissue obtained from HCC patients in which chronic hepatitis or cirrhosis is observed.

Furthermore, according to the present inventor's research, until now, DNA methylation levels of specific CpG sites in genomic DNA of liver tissues were detected by pyrosequencing, mass spectrometry, etc. A method has been proposed for assessing the risk of HCC in a patient by comparing it with a sample and a cutoff value to discriminate it (Patent Document 1). This method achieves highly sensitive and specific HCC risk assessment. However, there is a need for a method that can evaluate the risk of HCC more efficiently and at low cost while securing high sensitivity and specificity.

Recently, a method for detecting methylated DNA was disclosed by subjecting bisulfite-treated sample DNA to ion exchange chromatography (Patent Document 2). Furthermore, the present inventors have proposed a method of using ion-exchange chromatography on bisulfite-treated sample DNA to determine the prognosis of renal cell carcinoma based on the retention time using this principle ( Patent Document 3).

International Publication No. 2012/102377 International Publication No. 2014/136930 International Publication No. 2015/129916

A risk assessment method for hepatocellular carcinoma (HCC) based on detection of DNA methylation level using pyrosequencing etc. (for example, Patent Document 1) has high sensitivity and specificity, but it takes time to detect DNA methylation level. There is a drawback that it takes time and cost. The present invention provides a simple, rapid, low-cost method for evaluating HCC risk with high sensitivity and specificity.

The present inventors conveniently treat a sample DNA containing CpG sites obtained from liver tissue of a subject with bisulfite, amplify the DNA, and separate the amplification products by ion exchange chromatography. It has been found that DNA methylation of the CpG site can be detected rapidly. Furthermore, the present inventors found that the peak shape of the detection signal by the chromatography was different between the HCC high risk group and the low risk group. The peak shape of the detection signal by the chromatography serves as an index for risk assessment of HCC.

Even more surprisingly, the present inventors have identified other CpG sites when analyzing specific CpG sites in the risk assessment of HCC by DNA methylation analysis of CpG sites using the ion exchange chromatography described above. It has been found that significantly higher sensitivity and specificity can be achieved as compared to. Therefore, by combining DNA methylation analysis using the ion exchange chromatography with the use of the specific CpG site as a sample, HCC that combines simplicity and rapidity with high sensitivity and specificity. Risk assessment is realized.

Accordingly, the present invention provides the following.
[1] A method for evaluating the risk of hepatocellular carcinoma:
(1) a step of amplifying bisulfite-treated DNA from liver tissue of a subject, wherein the DNA comprises a CpG site in the exon region of the MGRN1 gene;
(2) a step of subjecting the obtained amplification product to ion exchange chromatography;
(3) determining whether or not the DNA is a DNA obtained from a subject having a high risk of developing hepatocellular carcinoma based on the shape of the peak of the detection signal of the chromatography;
Method, including.
[2] A method for evaluating the risk of hepatocellular carcinoma:
(1) a step of amplifying bisulfite-treated DNA from liver tissue of a subject, wherein the DNA comprises a CpG site in the exon region of the MGRN1 gene;
(2) a step of subjecting the obtained amplification product to ion exchange chromatography;
(3) determining whether or not the subject is at high risk of developing hepatocellular carcinoma based on the shape of the peak of the detection signal of the chromatography;
Method including.
[3] The method according to [1] or [2], wherein the DNA comprises a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence having at least 95% identity to the sequence.
[4] In (3) above, when the peak shape of the detection signal is a peak of methylated DNA having one peak, the DNA is selected as the DNA obtained from a subject having a high risk of developing hepatocellular carcinoma If the shape of the peak of the detection signal is bimodal, the DNA is selected as the DNA obtained from a subject having a low risk of developing hepatocellular carcinoma, any of [1] to [3] Or the method described in paragraph 1.
[5] The above (3) confirms that the peak of the methylated DNA is obtained by comparing the retention time of the detection signal peak with the retention time of the detection signal peak of the positive control or the negative control Including
Ion exchange chromatography of the DNA obtained when the detection signal of the positive control consists of the same sequence as the DNA from liver tissue of the subject and the DNA which is 100% methylated is treated with bisulfite and amplified It was obtained by
The ion exchange chromatography is performed on the DNA obtained when the detection signal of the negative control consists of the same sequence as the DNA from the liver tissue of the subject and the unmethylated DNA is bisulfite-treated and amplified. Obtained by
[4] The method described.
[6] The method according to any one of [1] to [5], wherein the ion exchange chromatography is anion exchange chromatography.

According to the present invention, while maintaining the sensitivity and specificity of detection as compared with the conventional method (for example, Patent Document 1) in which the DNA methylation level detected by pyrosequencing or the like is used as an indicator, The risk of hepatocellular carcinoma can be evaluated more simply and quickly.

Example of chromatogram obtained from Liv25 area | region of HCC patient origin non-cancer liver tissue sample (group N). Each figure represents data from an individual sample, and the symbols in each figure represent sample IDs. Plot of the first derivative of the data in FIG. An example of a chromatogram obtained from the Liv 25 region of a healthy liver tissue sample (group C). Each figure represents data from an individual sample, and the symbols in each figure represent sample IDs. Plot of the first derivative of the data in FIG.

As used herein, "hepatocellular carcinoma" (also referred to as Hepatocellular carcinoma; also referred to as HCC) means primary liver cancer that originates from hepatocytes that are the parenchyma of the liver.

As used herein, "risk of hepatocellular carcinoma" means the risk of developing hepatocellular carcinoma.

As used herein, the term "CpG site" refers to a site where phosphodiester bond (p) is established between cytosine (C) and guanine (G) on a DNA sequence.

As used herein, "DNA methylation" means that the carbon at position 5 of cytosine is methylated at the CpG site.

As used herein, the "methylation level" of DNA means the rate of methylation of the DNA (also referred to as the methylation rate).

As used herein, "at least 95% identity" with respect to a nucleotide sequence is 95% or more, preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, more preferably 99.5% This means the same as above.

In one embodiment, the present invention provides a method of assessing the risk of HCC comprising the following steps:
(1) a step of amplifying bisulfite-treated DNA from liver tissue of a subject, wherein the DNA comprises a CpG site in the exon region of the MGRN1 gene;
(2) a step of subjecting the obtained amplification product to ion exchange chromatography;
(3) A step of determining whether or not the DNA is a DNA obtained from a subject at high risk of developing HCC, based on the shape of the peak of the detection signal of the chromatography.

In another embodiment, the present invention provides a method of evaluating the risk of HCC, comprising the following steps:
(1) a step of amplifying bisulfite-treated DNA from liver tissue of a subject, wherein the DNA comprises a CpG site in the exon region of the MGRN1 gene;
(2) a step of subjecting the obtained amplification product to ion exchange chromatography;
(3) determining whether the subject is a subject at high risk of developing HCC, based on the shape of the peak of the detection signal of the chromatography.

In the present invention, DNA containing a CpG site of the exon region of the MGRN1 gene derived from liver tissue (also referred to as sample DNA in the following specification) obtained from a subject is treated with bisulfite and then amplified. Be done.

The “subject” in the present invention is not particularly limited. For example, healthy subjects, hepatitis B infected subjects, hepatitis C infected subjects, patients with chronic hepatitis, patients with liver cirrhosis, patients with hepatocellular carcinoma and those People who are suspected of Preferably, the subject is a human having hepatitis B, hepatitis C, chronic hepatitis or cirrhosis, which are generally known as those who are more likely to develop hepatocellular carcinoma.

There is no restriction | limiting in particular as a method to prepare "DNA derived from liver tissue" used by this invention, A well-known method can be selected suitably and can be used. As known methods for preparing genomic DNA, for example, phenol chloroform method (hepatic tissue is treated with proteinase K, surfactant (SDS), and phenol to denature proteins of the tissue, Next, a method of precipitating and extracting DNA from the tissue with chloroform, ethanol etc.), commercially available DNA extraction kit, eg, QIAamp DNA Mini kit (manufactured by Qiagen), Clean Columns (manufactured by NexTec), AquaPure (manufactured by Bio-Rad) And DNA extraction methods using ZR Plant / Seed DNA Kit (manufactured by Zymo Research), prepGEM (manufactured by ZyGEM), BuccalQuick (manufactured by TrimGen), and the like.

The liver tissue from which genomic DNA is prepared by such a method is not particularly limited. For example, intact liver tissue collected on biopsy etc., frozen liver tissue, formalin-fixed or paraffin-embedded liver tissue, etc. It can be mentioned. From the viewpoint of suppressing the degradation of genomic DNA in tissue, it is desirable to use frozen liver tissue. In the present invention, the condition of liver tissue (chronic hepatitis and cirrhosis stage, hepatitis virus infection, inflammation or fibrosis, etc.) used for preparation of genomic DNA and the distance from the focus of hepatocellular carcinoma are not particularly limited.

The “CpG site of the exon region of the MGRN1 gene” in the present invention refers to human MGRN1 (mahogunin ring finger 1) represented by NCBI Gene ID: 23295 ([www.ncbi.nlm.nih.gov/gene/23295]). A CpG site in the exon region of a gene.

The sample DNA used in the present invention is a DNA containing a CpG site of the exon region of the MGRN1 gene. For example, the sample DNA is a DNA containing a CpG site located in the upstream region of the MGRN1 gene exon. An example of such a sample DNA is a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1. Alternatively, a DNA consisting of a nucleotide sequence derived from the exon region of the MGRN1 gene and having at least 95% identity to the nucleotide sequence shown in SEQ ID NO: 1 is also an example of the sample DNA of the present invention. Preferably, the sample DNA comprises DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 95% identity with said sequence, more preferably consisting of the nucleotide sequence shown in SEQ ID NO: 1 It is DNA.

There is no restriction | limiting in particular in the method of a bisulfite process of sample DNA, A well-known method can be selected suitably and can be used. As a known method for bisulfite treatment, for example, EpiTect Bisulfite Kit (48) (manufactured by Qiagen) described later, MethylEasy (manufactured by Human Genetic Signatures Pty), Cells-to-CpG Bisulfite Conversion Kit (Applied) Examples include methods using commercially available kits such as Biosystems) and CpGenome Turbo Bisulfite Modification Kit (MERCK MILLIPORE).

As a method for amplifying the bisulfite-treated sample DNA, any nucleic acid amplification method such as PCR can be mentioned, and it is not particularly limited. The conditions for the amplification reaction are also not particularly limited, and known methods can be appropriately selected and used according to the sequence, length, amount, etc. of the DNA to be amplified. Preferably, the DNA is amplified by PCR. The chain length of the amplification product can be appropriately adjusted in consideration of factors such as shortening of the amplification reaction time, shortening of the analysis time in ion exchange chromatography, and maintenance of separation performance. For example, the length of the PCR amplification product is preferably 1000 bp or less, more preferably 500 bp or less, and still more preferably 300 bp or less. On the other hand, since the primer chain length for PCR is preferably 15 mer or more in order to avoid nonspecific amplification, the chain length of the PCR amplification product is preferably 31 bp or more, and more preferably 40 bp or more.

Preferred examples of the primer set for PCR amplification of DNA in the present invention include the primer sets shown in SEQ ID NOS: 4 and 5. Preferred examples of the conditions for the PCR are [94 ° C. 1 min → 64 ° C. 1 min → 72 ° C. 1 min] × 5 cycles → [94 ° C. 1 min → 62 ° C. 1 min → 72 ° C. 1 min] × 5 cycles → 94 ° C. 1 minute → 60 ° C. 1 minute → 72 ° C. 1 minute × 5 cycles → [94 ° C. 1 minute → 58 ° C. 1 minute → 72 ° C. 1 minute] × 35 cycles Absent.

Subsequently, in the present invention, the amplification product of the bisulfite-treated sample DNA obtained in the above amplification step is subjected to ion exchange chromatography. The ion exchange chromatography to be carried out in the present invention is preferably anion exchange chromatography. The packing material of the column used in the ion exchange chromatography performed in the present invention is not particularly limited as long as it is a substrate particle having a strong cationic group on the surface, but the surface shown in WO 2012/108516 Preferred are substrate particles having both a strong cationic group and a weak cationic group.

In the present specification, a strong cationic group means a cationic group which dissociates in a wide range of pH from 1 to 14. That is, the strong cationic group can be kept in a dissociated (cationized) state without being affected by the pH of the aqueous solution.

Examples of the strong cationic group include quaternary ammonium groups. Specific examples thereof include trialkyl ammonium groups such as trimethyl ammonium group, triethyl ammonium group, and dimethyl ethyl ammonium group. Moreover, as a counter ion of the said strong cationic group, halide ions, such as a chloride ion, a bromide ion, an iodide ion, are mentioned, for example.

The amount of the strong cationic group present on the surface of the substrate particle is not particularly limited, but the preferable lower limit per dry weight of the filler is 1 μeq / g, and the preferable upper limit is 500 μeq / g. If the amount of the strong cationic group is less than 1 μeq / g, the holding power may be weak and the separation performance may be deteriorated. When the amount of the strong cationic group exceeds 500 μeq / g, the retention strength becomes too strong to elute the DNA easily, which may cause problems such as too long analysis time.

In the present specification, a weak cationic group means a cationic group having a pka of 8 or more. That is, the weak cationic group is affected by the pH of the aqueous solution to change the dissociation state. That is, when the pH is higher than 8, the proton of the weak cationic group is dissociated, and the proportion without positive charge increases. Conversely, when the pH is lower than 8, the weak cationic group is protonated and the proportion with a positive charge increases.

As the said weak cationic group, a tertiary amino group, a secondary amino group, a primary amino group etc. are mentioned, for example. Among them, a tertiary amino group is desirable.

The amount of the weak cationic group present on the surface of the substrate particle is not particularly limited, but the preferable lower limit per dry weight of the filler is 0.5 μeq / g, and the preferable upper limit is 500 μeq / g. When the amount of the weak cationic group is less than 0.5 μeq / g, the separation performance may not be improved because the amount is too small. When the amount of the weak cationic group exceeds 500 μeq / g, similar to the strong cationic group, the coercivity becomes too strong and the DNA can not be easily eluted, causing problems such as too long analysis time. There is.

The amount of the strong cationic group or weak cationic group on the surface of the substrate particle can be measured by quantifying the nitrogen atom contained in the group. As a method of quantifying nitrogen, for example, Kjeldahl method can be mentioned. For example, in the case of a substrate particle having a strong cationic group and a weak cationic group, first, nitrogen contained in the strong cationic group is quantified after polymerization of the hydrophobic crosslinked polymer and the strong cationic group. Next, a weak cationic group is introduced into the polymer, and the total amount of nitrogen contained in the strong cationic group and the weak cationic group is quantified. The amount of nitrogen contained in the weak cationic group can be calculated from the determined value. Thus, based on the quantitative value of the nitrogen atom of the group, the amount of the strong cationic group and the amount of the weak cationic group contained in the filler can be adjusted within the above range.

As the base material particles, for example, synthetic polymer fine particles obtained by using a polymerizable monomer or the like, inorganic fine particles such as silica based particles, etc. can be used, but a hydrophobic cross-linked polymer made of a synthetic organic polymer Preferably it is a particle.

The hydrophobic cross-linked polymer is a hydrophobic cross-linked polymer obtained by copolymerizing at least one hydrophobic cross-linkable monomer and a monomer having at least one reactive functional group, at least one type of hydrophobic cross-linked polymer Any hydrophobic crosslinked polymer obtained by copolymerizing a hydrophobic crosslinking monomer, a monomer having at least one reactive functional group, and at least one hydrophobic non-crosslinkable monomer It may be.

The hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one monomer molecule, and, for example, ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) Di (meth) acrylic acid esters such as acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, tri (meth) acrylates such as trimethylolmethane tri (meth) acrylate and tetramethylol methane tri (meth) acrylate Acrylic acid esters or tetra (meth) acrylic acid esters, or aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene and the like can be mentioned. In the present specification, the above (meth) acrylate means acrylate or methacrylate, and (meth) acrylic means acrylic or methacrylic.

Examples of the monomer having a reactive functional group include glycidyl (meth) acrylate and isocyanate ethyl (meth) acrylate.

The hydrophobic non-crosslinkable monomer is not particularly limited as long as it is a non-crosslinkable polymerizable organic monomer having a hydrophobic property, and, for example, methyl (meth) acrylate, ethyl (meth) acrylate, Examples thereof include (meth) acrylic acid esters such as butyl (meth) acrylate and t-butyl (meth) acrylate, and styrenic monomers such as styrene and methyl styrene.

When the hydrophobic cross-linked polymer is obtained by copolymerizing the hydrophobic cross-linkable monomer and the monomer having the reactive functional group, the hydrophobic cross-linked polymer in the hydrophobic cross-linked polymer The lower limit of the content of the segment derived from the crosslinkable monomer is preferably 10% by weight, and more preferably 20% by weight.

The filler for ion exchange chromatography used in the present invention preferably has a polymer layer having the strong cationic group and the weak cationic group on the surface of the base particle. In the polymer having the strong cationic group and the weak cationic group, the strong cationic group and the weak cationic group are preferably derived from monomers independent of each other. Specifically, the filler for ion exchange chromatography used in the present invention is hydrophilic having the hydrophobic crosslinked polymer particles and the strong cationic group copolymerized on the surface of the hydrophobic crosslinked polymer particles. It is preferable that the weak cationic group is introduced onto the surface of a coated polymer particle comprising a polymer layer.

The hydrophilic polymer having the strong cationic group is composed of a hydrophilic monomer having the strong cationic group, and it is preferable to use a hydrophilic monomer having one or more kinds of the strong cationic group. It may contain the segment from which it originates. That is, as a method of producing a hydrophilic polymer having the strong cationic group, a method of polymerizing a hydrophilic monomer having the strong cationic group alone, having two or more strong cationic groups The method of copolymerizing a hydrophilic monomer, the method of copolymerizing the hydrophilic monomer which has this strong cationic group, and the hydrophilic monomer which does not have this strong cationic group, etc. are mentioned.

The hydrophilic monomer having a strong cationic group is preferably one having a quaternary ammonium group. Specifically, for example, ethyltrimethylammonium methacrylate, ethyltriethylammonium chloride methacrylate, ethyldimethylethylammonium chloride methacrylate, ethyldimethylbenzylammonium chloride methacrylate, ethyldimethylbenzylammonium chloride acrylate, ethyltrimethylammonium acrylate Chloride, ethyl triethyl ammonium chloride of acrylic acid, ethyl dimethyl ethyl ammonium chloride of acrylic acid, acrylamidoethyl trimethyl ammonium chloride, acrylamido ethyl triethyl ammonium chloride, acrylamido ethyl dimethyl ethyl ammonium chloride, etc. may be mentioned.

As a method for forming a layer of the hydrophilic polymer having the strong cationic group on the surface of the hydrophobic crosslinked polymer, a hydrophilic monomer having the strong cationic group on the surface of the hydrophobic crosslinked polymer The method of making it copolymerize is mentioned.

A well-known method can be used as a method of introduce | transducing a weak cationic group to the surface of the said coating polymer particle. Specifically, for example, as a method of introducing a tertiary amino group as the weak cationic group, the strong cationic property is obtained on the surface of a hydrophobic crosslinked polymer particle having a segment derived from a monomer having a glycidyl group. A method of copolymerizing a hydrophilic monomer having a group, and then reacting a reagent having a tertiary amino group with the glycidyl group; hydrophobic crosslinked polymer particles having a segment derived from a monomer having an isocyanate group A method of copolymerizing a hydrophilic monomer having the strong cationic group on the surface, and then reacting a reagent having a tertiary amino group with the isocyanate group; the strong on the surface of the hydrophobic crosslinked polymer particle A method of copolymerizing a hydrophilic monomer having a cationic group and a monomer having a tertiary amino group; using the silane coupling agent having a tertiary amino group to form the strong cationic group A method of introducing a tertiary amino group on the surface of a coated polymer particle having a layer of a hydrophilic polymer to be dispersed; said hydrophobic crosslinked polymer particle having a segment derived from a monomer having a carboxy group; Method of copolymerizing a hydrophilic monomer having a cationic group and then condensing the carboxy group and a reagent having a tertiary amino group using a carbodiimide; a segment derived from a monomer having an ester bond Copolymerizing the hydrophilic monomer having the strong cationic group on the surface of the hydrophobic cross-linked polymer particle having the above, hydrolyzing the ester bond portion, and then forming the carboxy group and 3 generated by the hydrolysis. The method of condensing with the reagent which has a class amino group using carbodiimide, etc. are mentioned. Among them, a hydrophilic monomer having the strong cationic group is copolymerized on the surface of a hydrophobic crosslinked polymer particle having a segment derived from a monomer having a glycidyl group, and then, a tertiary glycidyl group is obtained. A method of reacting a reagent having an amino group, or copolymerizing a hydrophilic monomer having the strong cationic group on the surface of a hydrophobic crosslinked polymer particle having a segment derived from a monomer having an isocyanate group, Next, a method of reacting a reagent having a tertiary amino group with the isocyanate group is preferable.

As a reagent having a tertiary amino group to be reacted with a reactive functional group such as a glycidyl group or an isocyanate group, a reagent having a tertiary amino group and a functional group capable of reacting to the reactive functional group is particularly preferable. It is not limited. As a functional group which can react to the said reactive functional group, a primary amino group, a hydroxyl group, etc. are mentioned, for example. Among them, a group having a primary amino group at the end is preferable. Specific examples of the reagent having a tertiary amino group having the functional group include N, N-dimethylaminomethylamine, N, N-dimethylaminoethylamine, N, N-dimethylaminopropylamine, and N, N-dimethylamino Butylamine, N, N-diethylaminoethylamine, N, N-diethylaminopropylamine, N, N-diethylaminobutylamine, N, N-diethylaminopentylamine, N, N-diethylaminohexylamine, N, N-dipropylaminobutylamine, N , N-dibutylaminopropylamine and the like.

The relative positional relationship between the strong cationic group, preferably a quaternary ammonium salt, and the weak cationic group, preferably a tertiary amino group, indicates that the strong cationic group is a substrate based on the weak cationic group. It is preferred to be at a position far from the surface of the particles, ie outside. For example, the weak cationic group is preferably within 30 Å from the surface of the base particle, and the strong cationic group is preferably within 300 Å from the surface of the base particle and outside the weak cationic group.

The average particle diameter of the substrate particles used for the filler for ion exchange chromatography used in the present invention is not particularly limited, but a preferable lower limit is 0.1 μm and a preferable upper limit is 20 μm. If the average particle size is less than 0.1 μm, the pressure in the column may be too high to cause separation failure. If the average particle size exceeds 20 μm, the dead volume in the column may be too large to cause separation failure. In addition, in this specification, an average particle diameter shows a volume average particle diameter, and it can measure using a particle size distribution measuring apparatus (made by AccuSizer 780 / Particle Sizing Systems etc.).

The amount of sample injection into the ion exchange chromatography column is not particularly limited, and may be appropriately adjusted according to the ion exchange capacity of the column and the sample concentration. The flow rate is preferably 0.1 mL / min to 3.0 mL / min, more preferably 0.5 mL / min to 1.5 mL / min. If the flow rate is low, improvement in separation can be expected, but if it is too low, analysis may take a long time, and the separation performance may be degraded due to broadening of peaks. On the other hand, if the flow rate is faster, there is an advantage in terms of shortening the analysis time, but the peaks are compressed, leading to a decrease in separation performance. Therefore, although it is a parameter suitably adjusted by the performance of a column, it is desirable to set to the range of the said flow rate. The retention time of each sample can be predetermined by performing a preliminary experiment for each sample. Although a known liquid transfer method such as a linear gradient elution method or a stepwise elution method can be used as a liquid transfer method, a linear gradient elution method is preferable as a liquid transfer method in the present invention. The size of the gradient (gradient) is adjusted according to the separation performance of the column and the characteristics of the analyte (here, the amplification product DNA obtained in the above amplification step) in the range of 0% to 100% of the eluent used for elution. It may be adjusted appropriately.

Well-known conditions can be used as a composition of the eluent used for the ion exchange chromatography implemented by this invention.

As a buffer used for the said eluant, it is preferable to use buffer containing known salt compounds and organic solvents. Specifically, for example, Tris-HCl buffer, TE buffer consisting of Tris and EDTA, TBA buffer consisting of Tris, boric acid and EDTA, and the like can be mentioned.

The pH of the eluent is not particularly limited, but the preferable lower limit is 5, and the preferable upper limit is 10. By setting this range, it is considered that the weak cationic group also effectively functions as an ion exchange group (anion exchange group). A more preferred lower limit to the pH of the eluent is 6, and a more preferred upper limit is 9.

As a salt contained in the said eluant, the salt which consists of halides, such as sodium chloride, potassium chloride, sodium bromide, potassium bromide, and an alkali metal, for example; Calcium chloride, calcium bromide, magnesium chloride, magnesium bromide Salts of halides and alkaline earth metals such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate and the like can be used. In addition, organic acid salts such as sodium acetate, potassium acetate, sodium succinate and potassium succinate can also be used. The salts may be used alone or in combination.

The salt concentration of the eluent may be appropriately adjusted according to the analysis conditions, but the lower limit is preferably 10 mmol / L, the upper limit is preferably 2000 mmol / L, and the lower limit is preferably 100 mmol / L, more preferably 1500 mmol / L. L

Furthermore, the eluent contains antichaotropic ions to further enhance the separation performance. Anti-chaotropic ions have properties opposite to that of chaotopic ions and function to stabilize the hydrated structure. Therefore, it has the effect of enhancing the hydrophobic interaction between the filler and the nucleic acid molecule. The main interaction of ion exchange chromatography used in the present invention is electrostatic interaction, but in addition, separation performance is enhanced by utilizing the action of hydrophobic interaction.

As an anti-chaotropic ion contained in the said eluant, phosphate ion (PO ₄ ^3- ), sulfate ion (SO ₄ ^2- ), ammonium ion (NH ₄ ⁺ ), potassium ion (K ⁺ ), sodium ion (Na ⁺ ) ⁺ ) Etc. Among the combinations of these ions, sulfate ions and ammonium ions are preferably used. The antichaotropic ions may be used alone or in combination. In addition, the component of the salt and buffer solution which are contained in the said elution solution may be contained in a part of the said antichaotropic ion. Such a component is suitable for the present invention because it has both the property as a salt or buffering capacity contained in the eluent and the property as an antichaotropic ion.

The concentration of the antichaotropic ion contained in the eluent may be appropriately adjusted according to the analyte, but is preferably 2000 mmol / L or less as the antichaotropic salt. Specifically, there can be mentioned a method in which the concentration of the antichaotropic salt is gradient-eluted in the range of 0 to 2000 mmol / L. Therefore, the concentration of the antichaotropic salt at the start of the chromatographic analysis does not have to be 0 mmol / L, and the concentration of the antichaotropic salt does not have to be 2000 mmol / L at the end of the analysis. The gradient elution method may be a low pressure gradient method or a high pressure gradient method, but a method of elution while performing precise concentration adjustment by a high pressure gradient method is preferable.

The anti-chaotropic ion may be added to only one of the eluents used for elution, or may be added to a plurality of eluents. The anti-chaotropic ion may also have the functions of both an effect of enhancing hydrophobic interaction between the packing agent and the DNA or a buffering capacity, and an effect of eluting the amplification product DNA from the column.

In the ion exchange chromatography used in the present invention, the column temperature when analyzing the amplification product DNA obtained in the above amplification step is preferably 30 ° C. or more, more preferably 40 ° C. or more, and still more preferably 45 It is preferably at least 60 ° C., more preferably at least 60 ° C. When the column temperature is less than 30 ° C., hydrophobic interactions between the packing material and the amplification product DNA become weak, and it becomes difficult to obtain a desired separation effect. On the other hand, when the column temperature of ion exchange chromatography is higher, amplification products derived from methylated DNA and amplification products derived from non-methylated DNA are more clearly separated. When the column temperature is 60 ° C. or higher, the difference in retention time of the peaks of the chromatographic detection signal between the amplification product derived from methylated DNA and the amplification product derived from non-methylated DNA is broadened, and each peak is more distinct. As a result, more accurate detection of DNA methylation and assessment of HCC risk become possible.

On the other hand, when the column temperature of ion exchange chromatography is 90 ° C. or higher, the double strand of the amplification product DNA is separated, which is not preferable for analysis. Furthermore, if the column temperature is 100 ° C. or higher, boiling of the eluent may occur, which is not preferable for analysis. Therefore, in the ion exchange chromatography used in the present invention, the column temperature when analyzing the amplification product DNA may be 30 ° C. or more and less than 90 ° C., preferably 40 ° C. or more and less than 90 ° C., more preferably 45 ° C to less than 90 ° C, more preferably 55 ° C to less than 90 ° C, still more preferably 60 ° C to less than 90 ° C, still more preferably 55 ° C to 85 ° C, still more preferably 60 ° C or more 85 ° C. or less.

Methylation of CpG sites in the exon region of the MGRN1 gene can be used as an index for evaluating the risk of HCC (Patent Document 1; JP 2010-148426 A; Int J Cancer, 2009, 125, 2854- 2862; Int J Cancer, 2011, 129, 1170-1179). Therefore, the risk of HCC can be evaluated based on the methylation level of sample DNA containing CpG sites in the exon region of the MGRN1 gene. In the present invention, the methylation level of the sample DNA appears as the difference in peak shape of the detection signal obtained by the above-mentioned ion exchange chromatography analysis.

When bisulfite treatment of DNA is performed, unmethylated BR> V tocin in the DNA is converted to uracil, but methylated cytosine remains cytosine. When the bisulfite-treated DNA is amplified, uracil derived from unmethylated cytosine is further replaced with thymine, resulting in a difference in the abundance ratio of cytosine to thymine between methylated DNA and unmethylated DNA. Therefore, the amplified DNA has different sequences depending on the methylation. When the DNA having the different sequence is subjected to ion exchange chromatography, a chromatogram showing different signals according to the difference in the sequence is obtained.

More specifically, in the above ion exchange chromatography analysis, the high level of DNA methylation is reflected in the retention time of the peak of the detection signal. For example, in the ion exchange chromatography analysis, 100% methylated DNA and unmethylated DNA can be detected as independent peaks, and typically the peak of methylated DNA is of unmethylated DNA. It appears at a retention time shorter than the peak (see Patent Document 2). Therefore, when methylated DNA is not present in the sample DNA or the amount thereof is a trace amount, the peak of the detection signal of the chromatography is in the form of one peak in which only the peak of unmethylated DNA appears. On the other hand, when the proportion of methylated DNA in the sample DNA is increased, a peak of methylated DNA appears separately at a shorter retention time, and the detection signal includes the peak of methylated DNA and the peak of unmethylated DNA. It has a bimodal shape. At this time, if the proportion of methylated DNA in the sample DNA is low, the peak of the second peak methylated DNA can appear as a bulge or a shoulder on the slope of the peak of the first peak unmethylated DNA. As the proportion of methylated DNA further increases, a second peak can clearly appear. Further, when the proportion of methylated DNA increases and methylated DNA dominates, the peak of the first peak becomes smaller and may appear as a bulge or a shoulder on the slope of the peak of the second peak. Furthermore, when the methylation of DNA proceeds and the sample DNA is almost completely methylated, the peak of the detection signal is in the form of one peak in which only the peak of methylated DNA appears. Thus, the peak shape of the signal obtained by ion exchange chromatography analysis of the sample DNA represents the methylation level of the sample DNA.

The peak shape of the signal obtained in the chromatographic analysis of the sample DNA reflects its DNA methylation level and has relevance to the risk of HCC. Therefore, based on the shape of the peak of the detection signal of ion exchange chromatography according to the present invention, the risk of developing HCC in a subject can be evaluated. The more components of the peak of methylated DNA in the peak of the detection signal, the more the canceration progresses in liver tissue, and the higher the risk of HCC in the subject. On the other hand, the more components of the peak of unmethylated DNA among the peaks of the detection signal, the less the canceration progresses in liver tissue, and the lower the risk of HCC in the subject.

In a preferred embodiment of the present invention, it is determined whether the shape of the peak of the detection signal of the ion exchange chromatography is unimodal or bimodal. Here, “one peak” means that the peak of methylated DNA appears as a peak of one peak. The determination of whether the shape of the peak is monomodal or bimodal may be determined from the shape of the curve where the detection signal is plotted against the retention time, or for a more precise analysis You may judge based on the derivative value of a detection signal. For example, when the first derivative of the detection signal is plotted against the retention time, the shape of the peak of the detection signal is determined to be bimodal when represented by a curve having two or more local maxima Ru.

In one embodiment of the present invention, based on the shape of the peak of the detection signal of the chromatography, it is determined whether the sample DNA is DNA obtained from a subject at high risk of developing HCC. Preferably, when the peak is one peak, the sample DNA is determined to be DNA obtained from a subject at high risk of developing HCC, and when the peak is bimodal, the sample DNA is HCC. It is determined that the DNA is obtained from a subject with a low risk of onset.

In another embodiment of the present invention, it is determined whether the subject is a subject at high risk of developing HCC based on the shape of the peak of the detection signal of the chromatography. Preferably, when the peak is unimodal, the subject is determined to be a subject at high risk of developing HCC, and when the peak is bimodal, the subject is at low risk of developing HCC It is determined to be a body.

As a method of determining the presence or absence of the peak of the detection signal by the chromatography, existing data processing software, such as LCsolution (Shimadzu Corporation), GRAMS / AI (Thermo Fisher Scientific), Igor Pro (WaveMetrics), etc. The peak detection used is mentioned. The determination method of the presence or absence of the peak using LCsolution is illustrated. Specifically, first, a section of retention time for which a peak is to be detected is designated. Next, various parameters are set in order to remove unnecessary peaks such as noise. For example, making the parameter "WIDTH" larger than the half width of the unnecessary peak, making the parameter "SLOPE" larger than the rising slope of the unnecessary peak, changing the setting of the parameter "DRIFT" For example, it is possible to select division or baseline division. Since different chromatograms can be obtained depending on the analysis conditions, the type and amount of DNA to be analyzed, etc., appropriate values may be set for the values of the parameters according to the chromatogram.

The derivative of the detected signal can be calculated automatically with the data processing software described above. Alternatively, the derivative value can be calculated by spreadsheet software (for example, Microsoft (registered trademark) Excel (registered trademark)) or the like.

The time range for calculating the differential value of the detection signal of chromatography can be set as appropriate. Moreover, the time range which detects the maximum value of a primary differential value can also be set suitably. For example, the maximum value of the first derivative can be detected in the time range from the rise to the end of the peak curve of the detection signal.

In the present invention, the chromatographic detection signal for the sample DNA can be compared to the detection signal for the control DNA. As control DNA, DNA having the same sequence as sample DNA and having a known methylation level (eg, 0% or 100%) is used.

For example, the detection signal for the 0% methylation control (negative control) is bisulfite treatment, nucleic acid amplification, and ion exchange using the same procedure as the sample DNA but using non-methylated DNA as described above. It can be obtained by performing chromatography. Also for example, the detection signal for the 100% methylation control (positive control) is bisulfite treatment, nucleic acid amplification, and in the above-described procedure using DNA which is identical in sequence to the sample DNA and 100% methylated. It can be obtained by performing ion exchange chromatography. Alternatively, the detection signals for the negative and positive controls are bisulfite-treated and nucleic acid amplified sample DNA with a methylation rate of 0% and 100%, respectively, which are then synthesized It can be obtained by subjecting to ion exchange chromatography.

Unmethylated DNA contained in the sample DNA appears as a peak with a retention time equivalent to that of the negative control. On the other hand, the retention time of the peak derived from methylated DNA contained in the sample DNA should shift toward the peak of the positive control, depending on the level of methylation. By comparing the retention time of the peak of the sample DNA with the negative control or the positive control, it can be confirmed that the signal from the sample DNA containing unmethylated DNA or methylated DNA has been correctly obtained. This procedure may be effective for preventing determination errors due to manipulation errors in sample DNA preparation and subsequent processing steps. However, this procedure is not always necessary, especially when a clear monomodal or bimodal peak is obtained from sample DNA.

Since the method for evaluating the risk of HCC according to the present invention uses ion exchange chromatography for detection of DNA methylation, it is compared with a conventional method for detecting DNA methylation by pyrosequencing or the like (for example, Patent Document 1). It is a very simple and quick method. Furthermore, surprisingly, in the present invention, the methylation of CpG sites in the exon region of the MGRN1 gene was used as an index, which made it possible to evaluate the risk of HCC with extremely high sensitivity and specificity. As shown in the examples described below, in the risk assessment of HCC using detection of DNA methylation by ion exchange chromatography, DNA (Liv 25 region) containing CpG sites in the exon region of the MGRN1 gene as target DNA for detecting methylation. When SEQ ID NO: 1) is selected, DNA (Liv27 region; SEQ ID NO: 2) containing the CpG site in the vicinity (CpG site of intron region of MGRN1 gene) or DNA containing the CpG site of different gene (KDM4B) (Liv28 Compared to the region; SEQ ID NO: 3), the sensitivity and specificity were significantly improved. The CpG site in the vicinity and the CpG site of the KDM4B gene are the sites achieving 100% sensitivity and specificity in HCC risk assessment by pyrosequencing (see Patent Document 1, Table 4). However, both of these two sites greatly reduced the sensitivity or specificity as assessed by ion exchange chromatography. On the other hand, the DNA containing the exon region CpG site of the MGRN1 gene achieved extremely high sensitivity and specificity equivalent to the method using pyrosequencing even in the HCC risk evaluation by ion exchange chromatography according to the present invention. Therefore, the risk assessment method for HCC according to the present invention is an excellent method that can combine rapidity, simplicity, extremely high sensitivity and specificity.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to the following examples.

〔Method〕
(1) Patients and Samples Patients with hepatitis virus (HBV or HCV) infection history, 36 non-cancerous liver tissues (group N) samples removed from patients with HCC, and patients without hepatitis virus infection history and HCC The samples were 36 healthy liver tissues (group C) samples removed from the

(2) Preparation of DNA High-molecular weight DNA was extracted by treating a fresh frozen tissue sample obtained from the patient with phenol-chloroform and then dialyzing (Sambrook, J. et al., Molecular cloning: Experimental manual) Third edition, Cold Spring Harbor Publishing, NY, pages 6.14-6.15). The resulting DNA500ng, using EZ DNA Methylation-Gold ^TM kit (Zymo manufactured Research), was subjected to bisulfite treatment.

(3) PCR amplification The bisulfite-treated genomic DNA obtained in (2) was PCR amplified. In PCR, the following three different regions containing CpG sites in genomic DNA were amplified:
Liv25 region: DNA region containing CpG site of exon region of MGRN1 gene Liv27 region: CpG site near Liv25 region (CpG site of intron region of MGRN1 gene) Liv28 region: CpG site of different gene (KDM4B) The DNA region amplified region (a sequence before bisulfite treatment) containing the DNA and the nucleotide sequence of the primer used for amplification are shown in Table 1. For each region, DNA of 0% methylation (negative control) and DNA of 100% methylation (positive control) of the same sequence were prepared and PCR amplified after bisulfite treatment in the same procedure.

Composition of PCR reaction solution (50 μL): template DNA 75 ng, 1 × QIAGEN PCR buffer (manufactured by QIAGEN), 200 μmol / L dNTP Mix (manufactured by TOYOBO), 2.5 U HotStart Taq Plus DNA Polymerase (manufactured by QIAGEN), 0. 2 μmol / L forward and reverse primers. The PCR conditions were as follows:
Liv25:
[94 ° C 1 minute → 64 ° C 1 minute → 72 ° C 1 minute] × 5 cycles → [94 ° C 1 minute → 62 ° C 1 minute → 72 ° C 1 minute] × 5 cycles → [94 ° C 1 minute → 60 ° C 1 minute → 72 ° C 1 minute] × 5 cycles → [94 ° C 1 minute → 58 ° C 1 minute → 72 ° C 1 minute] × 35 cycles Liv 27:
[94 ° C. 1 min → 56 ° C. 1 min → 72 ° C. 1 min] × 50 cycles Liv 28:
[94 ° C 1 minute → 56 ° C 1 minute → 72 ° C 1 minute] × 50 cycles After completion of the PCR, apply 5μL of the reaction solution mixed with 1μL of the loading dye solution to a 3% agarose gel to which ethidium bromide has been added beforehand. Electrophoresis was performed to confirm that the desired PCR amplification product was obtained.

(4) Preparation of Anion Exchange Column In 2000 mL of a 3% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.) aqueous solution in a reactor equipped with a stirrer, 200 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), triethylene glycol di A mixture of 100 g of methacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100 g of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.0 g of benzoyl peroxide (manufactured by Kishida Chemical Co., Ltd.) was added. The mixture was heated with stirring and polymerized at 80 ° C. for 1 hour under a nitrogen atmosphere. Next, 100 g of ethyltrimethyl ammonium methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in ion exchange water as a hydrophilic monomer having a strong cationic group. This was added to the same reactor and polymerized similarly at 80 ° C. for 2 hours under nitrogen atmosphere with stirring. The obtained polymerized composition was washed with water and acetone to obtain coated polymer particles having a layer of a hydrophilic polymer having a quaternary ammonium group on the surface. The obtained coated polymer particles were measured using a particle size distribution measuring device (AccuSizer 780 / Particle Sizing Systems), and the average particle size was 10 μm.

10 g of the obtained coated polymer particles were dispersed in 100 mL of ion exchanged water to prepare a pre-reaction slurry. Next, while the slurry was being stirred, 10 mL of N, N-dimethylaminopropylamine (manufactured by Wako Pure Chemical Industries, Ltd.), which is a reagent having a weak cationic group, was added and reacted at 70 ° C. for 4 hours. After completion of the reaction, the supernatant was removed using a centrifuge ("Himac CR20G" manufactured by Hitachi, Ltd.) and washed with ion-exchanged water. After washing, the supernatant was removed using a centrifuge. This washing with ion exchange water was further repeated four times to obtain a filler for ion exchange chromatography having quaternary ammonium groups and tertiary amino groups on the surface of the substrate particles.

The obtained packing material for ion exchange chromatography was packed in a stainless steel column (column size: inner diameter 4.6 mm × length 20 mm) of a liquid chromatography system.

(5) HPLC analysis Ion exchange chromatography was performed using the anion exchange column prepared in (4) under the following conditions to separate and detect each PCR amplification product obtained in (3).
System: LC-20A series (made by Shimadzu Corporation)
Eluent: Eluent A 25 mmol / L Tris-HCl buffer (pH 7.5)
Eluent B 25 mmol / L Tris-HCl buffer (pH 7.5)
+1 mol / L ammonium sulfate Analysis time: 15 minutes analysis time Elution method: The mixing ratio of eluent B was linearly increased by the following gradient conditions.
0 minutes (eluent B 40%) → 10 minutes (eluent B 100%)
Sample: PCR amplification product obtained in (2) Flow rate: 1.0 mL / min
Detection wavelength: 260 nm
Sample injection volume: 5 μL, 2 μL for negative control and positive control
Column temperature: 70 ° C

(6) HCC risk evaluation by methylation analysis of CpG site in MGRN1 gene exon region using chromatography DNA containing CpG site in the exon region of MGRN1 gene of HCC patient-derived non-cancer liver tissue sample (group N) (Liv 25 Among the HPLC chromatograms obtained from the region), a typical example is shown in FIG. The chromatograms of unmethylated DNA (negative control) and 100% methylated DNA (positive control) are also displayed superimposed on each figure of FIG. In these N group samples, only a monomodal peak appeared overlapping with the peak of the positive control. Also, the first derivative of these data (FIG. 2) was a curve having one maximum value. Samples that were not single peak, such as having a shoulder at the peak in the chromatogram, could be identified by the first derivative curve becoming a curve having two maximum values (not shown).

Among the HPLC chromatograms obtained from the Liv25 region of a healthy liver tissue sample (group C), a typical example is shown in FIG. The chromatograms of unmethylated DNA (negative control) and 100% methylated DNA (positive control) are also shown superimposed in FIG. In these group C samples, bimodal peaks appeared. From the comparison with the negative control and the positive control, it was estimated that these bimodal peaks include the peak of 100% methylated DNA and the peak of DNA with lower methylation rate. Also, the first derivative of these data (FIG. 4) was a curve having two maximum values.

As described above, ion exchange chromatography analysis was able to detect an increase in DNA methylation level of DNA (Liv25 region) containing CpG sites in the exon region of the MGRN1 gene in non-cancerous liver tissue of HCC patients. In addition, the shape of the peak of the chromatographic analysis for the Liv25 region was distinctly different between the N group and the C group, and based on the shape of the peak, it was possible to distinguish the tissue of HCC patients from the healthy tissue.

(7) HCC risk assessment by methylation analysis of other regions The HPLC chromatogram obtained from the Liv 27 region also tends to have a single peak in the N group and a bimodal peak in the C group, as in the Liv 25 region. It was done. On the other hand, HPLC chromatograms obtained from the Liv 28 region tended to have a bimodal peak in the N group and a single peak in the C group. However, in the Liv27 region and Liv28 region, there are many cases where peaks that do not match the above tendency are obtained in any of the N group and C group, and the shape of the HCC patient's tissue and healthy tissue can not be distinguished accurately It has been suggested.

(8) Inter-region comparison of sensitivity and specificity of HCC risk assessment The HCC risk assessment is performed using the chromatograms of Liv25, Liv27 and Liv28 regions, and the sensitivity (positive agreement rate) and specificity (negative agreement rate) Was calculated. In the Liv25 and Liv27 regions, a sample from which a chromatogram having a single peak was obtained was determined to be HCC risk positive, and a sample from which a chromatogram having a bimodal peak was obtained was determined to be HCC risk negative. In the Liv28 region, a sample for which a chromatogram having a bimodal peak was obtained was judged as positive for HCC risk, and a sample for which a chromatogram having a single peak was obtained was judged as negative for HCC risk. Table 2 shows the number of samples judged positive and negative for HCC risk in Groups N and C for each region, and the sensitivity and specificity of the evaluation. The evaluation in the Liv25 region was very high in both sensitivity and specificity, with significantly higher accuracy compared to the other regions.

Claims (6)

How to assess the risk of hepatocellular carcinoma:
(1) a step of amplifying bisulfite-treated DNA from liver tissue of a subject, wherein the DNA comprises a CpG site in the exon region of the MGRN1 gene;
(2) a step of subjecting the obtained amplification product to ion exchange chromatography;
(3) determining whether or not the DNA is a DNA obtained from a subject having a high risk of developing hepatocellular carcinoma based on the shape of the peak of the detection signal of the chromatography;
Method, including. How to assess the risk of hepatocellular carcinoma:
(1) a step of amplifying bisulfite-treated DNA from liver tissue of a subject, wherein the DNA comprises a CpG site in the exon region of the MGRN1 gene;
(2) a step of subjecting the obtained amplification product to ion exchange chromatography;
(3) determining whether or not the subject is at high risk of developing hepatocellular carcinoma based on the shape of the peak of the detection signal of the chromatography;
Method including. The method according to claim 1 or 2, wherein the DNA comprises a nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence having at least 95% identity to the sequence. In the above (3), when the peak shape of the detection signal is a peak of methylated DNA having one peak, the DNA is selected as DNA obtained from a subject having a high risk of developing hepatocellular carcinoma, The method according to any one of claims 1 to 3, wherein when the shape of the peak of the detection signal is bimodal, said DNA is selected as the DNA obtained from a subject having a low risk of developing hepatocellular carcinoma. Method. (3) confirms that the peak of the methylated DNA is obtained by comparing the retention time of the peak of the detection signal with the retention time of the peak of the detection signal of the positive control or the negative control Including
Ion exchange chromatography of the DNA obtained when the detection signal of the positive control consists of the same sequence as the DNA from liver tissue of the subject and the DNA which is 100% methylated is treated with bisulfite and amplified It was obtained by
The ion exchange chromatography is performed on the DNA obtained when the detection signal of the negative control consists of the same sequence as the DNA from the liver tissue of the subject and the unmethylated DNA is bisulfite-treated and amplified. Obtained by
The method of claim 4. The method according to any one of claims 1 to 5, wherein the ion exchange chromatography is anion exchange chromatography.

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